title: Online comprehensive RPLC × RPLC with mass spectrometry detection for the analysis of proteome samples.
abstract: LC-MS-based shotgun proteomics relies both on the power of the separation techniques and the sensitivity of detection methods. As a viable alternative to classical approaches in this field, we developed a fully automated, comprehensive 2D LC system, in which RPLC × RPLC was coupled to MS detection, for the first time, and applied for the analysis of tryptic digests obtained from α-casein and dephosphorylated α-casein. The use of a significantly different pH in the two dimensions allowed us to attain high peak capacity, despite the employment of novel identical stationary phases. Furthermore, such a combination addresses compatibility issues, thus allowing straightforward interfacing in online 2D LC configuration, as well as direct linkage to a mass spectrometer. A theoretical peak capacity of ca. 8500 was calculated for the setup, employing four serially coupled C18 columns in the first dimension (600 × 2.1 mm, 2.7 μm d.p.), operated under basic conditions, and 3 cm length of the same stationary phase (30 × 4.6 mm, 2.7 μm d.p. column), under acidic conditions, for fast second dimension analysis.
authors.affiliation: Dipartimento Farmaco-chimico, Facoltà di Farmacia, Università di Messina, viale Annunziata, 98168 Messina, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Donato
authors.forename: Francesco
authors.initials: F
authors.lastname: Cacciola
authors.forename: Eduardo
authors.initials: E
authors.lastname: Sommella
authors.forename: Chiara
authors.initials: C
authors.lastname: Fanali
authors.forename: Laura
authors.initials: L
authors.lastname: Dugo
authors.forename: Marina
authors.initials: M
authors.lastname: Dachà
authors.forename: Pietro
authors.initials: P
authors.lastname: Campiglia
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.forename: Paola
authors.initials: P
authors.lastname: Dugo
authors.forename: Luigi
authors.initials: L
authors.lastname: Mondello
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D009862:Online Systems; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D002364:Caseins; D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin